Methods and Systems for T Cell Expansion

ABSTRACT

The present disclosure provides a system for mimicking the secondary lymphoid organs where suspension cells (e.g., T cells) are expanded; methods of expanding, activating, and transfecting the suspension cells in the synthetic microenvironment, and suspension cells produced by such systems and methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/410,877, filed on 21 Oct. 2016, the disclosure of which is hereinincorporated by reference in its entirety.

GOVERNMENT SPONSORSHIP

This invention was made with government support under Grant Nos. 12567G5and 1547638 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND OF THE DISCLOSURE 1. Field

Embodiments of the present disclosure relate generally to methods andsystems for suspension cell (for example and not limitation, T cells)expansion, and more specifically to synthetic microenvironments capableof mimicking T cell niches within secondary lymphoid organs (such as forexample and not limitation, lymph nodes). For example and notlimitation, these synthetic microenvironments comprise functionalizedmacroporous three-dimensional (3D) microcarriers that contain antibodiesto enhance T cell expansion and activation, as well as optionally enabletransfection. T cells obtainable by these systems and methods includegamma-delta T cells as well as alpha-beta T cells, including for exampleand not limitation, recombinant T cells, gene modified T cells, chimericantigen receptor (CAR) T cells, unmodified T cells, and CCR7⁺CD62⁺central memory T cells.

2. Background

Immunotherapy using adoptive T cell transfer (ACT) is a highly promisingapproach in treating cancers, infectious and autoimmune diseases, aswell as for transplantation associated problems. In particular,anti-tumor ACT, using tumor-isolated or genetically-engineered T cells(e.g., Chimeric Antigen Receptor (CAR) T cells), has shown greatpotential in clinical trials of various cancers (1-3).

Physiological T cell expansion occurs primarily in secondary lymphoidorgans (e.g., lymph nodes (LNs), spleen, gut-associated lymphoid tissueetc.). Upon interaction with antigen presenting cells (APC, such as forexample and not limitation, dendritic cells) displaying foreign antigenepitopes, T cells engage with APCs through several surface moleculesincluding T cell receptors (TCRs), CD3, CD28, etc. T cells expand as aresult of this direct signaling aided by a set of locally secretedcytokines by themselves and APCs. The high cell density of the T cellzone in LNs ensures that cytokines are presented at high localconcentrations and efficient autocrine and paracrine signaling takesplace. Current T cell expansion methods using suspension cultures, istherefore non-physiological and requires very high dosage of cytokinesand do not recapitulate the cell-cell communication required forefficient T cell expansion. Even though T cells do form loose aggregatesin these suspension cultures, synthetic 3D niches that mimic a more LNlike high-density culture environment with efficient cell-cellcommunication and relevant extracellular-matrix, could significantlyimprove T cell expansion and quality.

Currently, the most common approach to T cell expansion is the use ofsoluble anti-CD3 or anti-CD3/anti-CD28 dynabeads with suspensioncultures. Although surface-immobilized antibodies (Abs), such as onmicrobeads, signal more robustly and mimic the APC/T-cell interactionsbetter, a key issue with bead-based expansion is that newly generatedcells (progenies) have minimal interaction with anti-CD3/anti-CD28 beadsand thus do not expand further.

In adults, each dose of CAR-T cell therapy requires approximately10⁸-10⁹ cells to be injected (4-6). Considering the additional number ofcells that are required for safety testing and quality control (e.g.,testing of sterility, endotoxin level, cell purity, particulateimpurity, stability, potency, etc.), the total number of cells for eachbatch of production must exceed ˜10⁹. Unfortunately, the number ofautologous T cells that can be harvested from cancer patients,especially those with advanced cancer and those undergoing radiation andchemotherapy could be very limited, which further decreasessignificantly in the process of genetic-modification (CAR transductionwith lentivirus). Current expansion protocols using suspension cultureswith large amounts of IL-2, IL-7 or IL-15 can only achieve 10-100-foldexpansion in 1-2 weeks of processing, thus generally resulting in justover a single dose of T cells from each batch with no option of multipledosing or storage. If multiple doses are to be administered from asingle bioprocess, even more cells would be necessary (especially giveninefficient freeze-thaw process). Furthermore, thetransduction/expansion process generally results in a heterogeneouspopulation of T cells with multiple phenotypes (effector cells, memorycells, exhausted cells, high or low cytokine-secreting cells etc.) andwhich of these phenotypes are most suited for maximal in vivo anti-tumorefficacy, is still largely unknown. Although for B cell malignancies,memory-type T cells (CD62L⁺CCR7⁺) have been shown to have the highestcorrelative potency, primarily due to their ability to survive in vivoand better home into the tumor site (lymph nodes); that has not beenestablished for other cancers (7). Thus, new bioprocess engineeringmethods to efficiently expand CAR-T populations, in terms of numbers,cell quality and potency, is critically needed to enable broad clinicaluse of this promising therapy.

Thus, despite its potential and recent success, current approaches forCAR-based ACT are severely constrained by (a) the limited availabilityof autologous T cells from cancer patients; (b) difficulty in robustlyand reproducibly expanding these cells to enough numbers for multipleadministrations; and (c) lack of methods that selectively expand themost potent sub-population of T cells for specific applications (forexample but not limitation, memory T cells and/or T cells with superiortransport properties). As a result, new cell-manufacturing concepts thatwould allow large scale production of therapeutic T cells, such as forexample and not limitation, therapeutic CAR-T cells, without losingtheir potency and safety, are needed.

What is needed, therefore, is a synthetic microenvironment capable ofmimicking T cell niches within secondary lymphoid organs such as forexample and not limitation, lymph nodes, the anatomical location wherenatural T cell activation and expansion take place in the body. Thesystems should take advantage of 3D microcarriers—which are widely usedfor adherent cells in industry practice but not for suspensioncells—that can be functionalized with antibodies to promote suspensioncell (e.g., T cell) activation and expansion. The systems should providesuspension cells with improved potency and efficacy and allow forspecific highly potent sub populations to expand selectively. It is tosuch systems and methods of producing suspension cells, such as forexample and not limitation, T cells, that embodiments of the presentdisclosure are directed.

BRIEF SUMMARY OF THE DISCLOSURE

As specified in the Background Section, there is a great need in the artto identify technologies for improved methods and systems forlarge-scale production of suspension cells, such as for example and notlimitation, T cells, and use this understanding to develop novelbioreactor systems and methods. The present disclosure satisfies thisand other needs. Embodiments of the present disclosure relate generallyto synthetic microenvironment capable of mimicking T cell niches withinsecondary lymphoid organs (such as for example and not limitation, lymphnodes) and more specifically to macroporous 3D microcarriers that can befunctionalized with antibodies to promote suspension cell (e.g., T cell)activation and expansion to mimic the environment found in lymph nodes.The system should provide suspension cells with improved potency andefficacy. It is to such systems and methods of producing suspensioncells, such as for example and not limitation, T cells, that embodimentsof the present disclosure are directed. T cells produced by the systemsand methods described herein include, but are not limited to,recombinant T cells, gene modified T cells, chimeric antigen receptor(CAR) T cells, unmodified T cells, and CCR7⁺CD62⁺ central memory Tcells.

The present disclosure provides a system for mimicking the secondarylymphoid organs where suspension cells (e.g., T cells) are expanded;methods of expanding, activating, and transfecting the suspension cellsin the synthetic microenvironment, and suspension cells produced by suchsystems and methods.

In one aspect, the disclosure provides a system for expanding,activating, and/or transfecting suspension cells comprising: a porousmicrocarrier; and the suspension cells.

In some embodiments, the suspension cells are isolated from a patient'sblood or organs, including both normal and/or diseased tissues.

In some embodiments, the suspension cells are T cells.

In other embodiments, the porous microcarrier is three dimensional. Insome embodiments, the porous microcarrier comprises proteins,carbohydrates, lipids or nucleic acids.

In some embodiments, the porous microcarrier comprises gelatin or otherextracellular matrix components.

In yet other embodiments, the porous microcarrier is functionalized. Insome embodiments, the functionalized porous microcarrier comprises atleast one of antibodies, aptamers, and phage-display identified peptideligands.

In some embodiments, the antibodies comprise antibodies that arespecific for the suspension cells. In other embodiments, the antibodiescomprise antibodies that are specific for T cells. In some embodiments,the antibodies comprise anti-CD2, anti-CD3 and/or anti-CD28 antibodies.

In an embodiment, the system in any of the preceding embodiments furthercomprises a bioreactor. In some embodiments, the bioreactor comprises aclosed bioreactor and an open bioreactor. In some embodiments, the openbioreactor comprises a static culture vessel with a gas-permeablebottom. In other embodiments, the closed bioreactor comprises astirred-type bioreactor, a bag bioreactor, and a perfusion bioreactor.

In another embodiment, the system in any of the preceding embodimentsfurther comprises at least one of culture medium, at least one cytokine,and/or at least one viral vector, and optionally at least one growthfactor.

In some embodiments, the at least one cytokine comprises IL2 andoptionally at least one of IL7 or IL15. In other embodiments, the atleast one viral vector is configured for use in gene therapy (such asfor example and not limitation, a lentiviral vector, a retroviralvector, an adenoviral vector, and an adeno-associated viral vector).

In any of the embodiments described herein, the suspension cells cancomprise recombinant T cells, gene modified T cells, chimeric antigenreceptor (CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ centralmemory T cells.

In some embodiments, the at least one viral vector comprises a CARtransgene and optionally at least one additional gene, wherein the atleast one additional gene comprises therapeutic genes, surface markergenes, reporter genes, suicide genes, chemokine receptor genes,cytokine-expressing genes, and/or immune-checkpoint receptor genes.

In any of the preceding embodiments, the porous microcarrier ismacroporous. In some embodiments, the porous microcarrier is degradable,such as for example and not limitation, biodegradable.

In a related aspect, the disclosure provides a method of expanding,activating, and/or transfecting suspension cells, the method comprising:obtaining a blood sample from a patient; isolating suspension cells fromthe blood sample; introducing the suspension cells to a bioreactorcomprising a porous microcarrier; activating the suspension cells;expanding the suspension cells; optionally transfecting the suspensioncells; preparing the suspension cells for transfusion into the patient;and transfusing the suspension cells into the patient.

In some embodiments, the blood or tissue sample is obtained byleukapharesis.

In other embodiments, the suspension cells are T cells.

In some embodiments, the step of isolating suspension cells from theblood sample further comprises bead separation or magnetic beadseparation.

In other embodiments, the bioreactor comprises a closed bioreactor andan open bioreactor. In some embodiments, the closed bioreactor comprisesa stirred-type bioreactor, a bag bioreactor, and a perfusion bioreactor.In other embodiments, the open bioreactor comprises a static culturevessel with a gas-permeable bottom.

In some embodiments, the porous microcarrier is three dimensional. Inother embodiments, the porous microcarrier comprises proteins,carbohydrates, lipids or nucleic acids. In still other embodiments, theporous microcarrier comprises gelatin or other extracellular matrixcomponents.

In some embodiments, the porous microcarrier is functionalized. In otherembodiments, the functionalized porous microcarrier comprises at leastone of antibodies, aptamers, and phage-display identified peptideligands. In still other embodiments, the antibodies comprise antibodiesthat are specific for the suspension cells. In some embodiments, theantibodies comprise antibodies that are specific for T cells. In someembodiments, the antibodies comprise anti-CD2, anti-CD3 and/or anti-CD28antibodies.

In an embodiment of any of the foregoing methods, the method furthercomprises at least one of culture medium, at least one cytokine, and/orat least one viral vector, and optionally at least one growth factor. Insome embodiments, the at least one cytokine comprises IL2, andoptionally at least one of IL7 or IL15.

In an embodiment of any of the foregoing methods, the activation stepfurther comprises agitation, optionally under hypoxic conditions. Insome embodiments, the agitation can be periodic or continuous. In otherembodiments, the activation step can last for at least one day, at leasttwo days, at least three days, and at least four days, and all ranges oftime in between.

In an embodiment of any of the foregoing methods, the expansion stepfurther comprises seed trains, optionally under hypoxic conditions. Forexample and not limitation, the expansion step can include increasingthe size of the flask and/or bioreactor as the culture grows, which canalso involve moving the expanding cells to a new vessel, and culturemedia exchanges every 2-3 days (particularly if a static culture). Theexpansion step can last for at least one day, at least two days, atleast three days, at least four days, at least five days, at least sixdays, at least seven days, at least eight days, at least nine days, atleast ten days, and at least eleven days, and all ranges of time inbetween.

In an embodiment of any of the foregoing methods, the optionaltransfection step further comprises adding at least one viral vector tothe bioreactor comprising the suspension cells.

In an embodiment of any of the foregoing methods, the suspension cellscomprise recombinant T cells, gene modified T cells, chimeric antigenreceptor (CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ centralmemory T cells.

In some embodiments, the at least one viral vector is configured for usein gene therapy. In other embodiments, the at least one viral vectorcomprises a CAR transgene and optionally at least one additional gene,wherein the at least one additional gene comprises therapeutic genes,surface marker genes, reporter genes, suicide genes, chemokine receptorgenes, cytokine-expressing genes, and/or immune-checkpoint receptorgenes.

In an embodiment of any of the foregoing methods, the preparation stepfurther comprises cell expansion and downstream bioprocessing. In someembodiments, the cell expansion and downstream bioprocessing comprisescell separation, purification, packaging, preservation, storage,shipping and transport, thawing, formulation, resuspension, andtransfusion.

In an embodiment of any of the foregoing methods, the transfusion stepfurther comprises injection, intravenous administration, andimplantation (such as for example and not limitation, implantation of asustained delivery device).

In an embodiment of any of the foregoing methods, the porousmicrocarrier is macroporous. In any of the preceding embodiments, theporous microcarrier is macroporous. In some embodiments, the porousmicrocarrier is degradable, such as for example and not limitation,biodegradable.

In a related aspect, the disclosure provides a T cell obtained from anyof the systems disclosed herein.

In a related aspect, the disclosure provides a T cell obtained from anyof the methods disclosed herein.

In one embodiment, the T cell is a memory cell. In another embodiment,the T cell is CCR7⁺CD62L⁺. In another embodiment, the T cell is acentral memory T cell. In yet another embodiment, the T cell is a CD4 Tcell or a CD8 T cell. In some embodiments, the T cell comprisesrecombinant T cells, gene modified T cells, chimeric antigen receptor(CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ central memory Tcells.

In a related aspect, the disclosure provides a composition comprising atleast one T cell as described herein. In some embodiments, thecomposition further comprises at least one carrier. In otherembodiments, the composition is formulated for intravenousadministration.

In a related aspect, the disclosure provides a pharmaceuticalcomposition comprising at least one T cell as described herein. In someembodiments, the pharmaceutical composition further comprises at leastone carrier. In other embodiments, the composition is formulated forintravenous administration. In some embodiments, the pharmaceuticalcomposition further comprises at least one additional therapeutic agent.

In a related aspect, the disclosure provides the use of a composition(including a pharmaceutical composition) as described herein to treat adisease or condition in a patient in need thereof. In some embodiments,the disease or condition comprises genetic diseases, cancers,infections, autoimmune diseases, and/or transplant complications. Insome embodiments, the use further comprises a second therapeutic methodor agent, such as for example and not limitation, a cancer drug, animmunotherapeutic, an immunosuppressant, an autoimmune therapeuticagent, and/or a therapeutic for treating infections.

In a related aspect, the disclosure provides method of treating adisease or condition in a patient in need thereof, comprisingadministering a composition (including a pharmaceutical composition) asdescribed herein to said patient. In some embodiments, the disease orcondition comprises genetic diseases, cancers, infections, autoimmunediseases, and/or transplant complications. In some embodiments, thetreatment further comprises a second therapeutic method or agent, suchas for example and not limitation, a cancer drug, an immunotherapeutic,an immunosuppressant, an autoimmune therapeutic agent, and/or atherapeutic for treating infections.

In an embodiment of any of the foregoing systems, the porousmicrocarrier is configured for activation, expansion, and/ortransfection of the suspension cells.

In an embodiment of any of the foregoing methods, the porousmicrocarrier is configured for activation, expansion, and/ortransfection of the suspension cells.

These and other objects, features and advantages of the presentdisclosure will become more apparent upon reading the followingspecification in conjunction with the accompanying description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIGS. 1A-1B. Optimization of streptavidin coating of microcarriers. FIG.1A) Streptavidin surface density as a function of sulfo-NHS-biotin addedper mass of microcarrier for Cultispher S and Cultispher Gmicrocarriers. FIG. 1B) Binding site density as a function ofsulfo-NHS-biotin amount added per mass of Cultispher S (CuS)microcarriers.

FIGS. 2A-2B. Verification of open biotin binding sites. Zeiss Lightsheetmicroscope imaging of (FIG. 2A) uncoated CuS microcarriers and (FIG. 2B)CuS microcarriers optimally coated with streptavidin and saturated withFITC-biotin.

FIG. 3. Antibody coating density as function of CD3/CD28 percentage.Microcarriers coated with streptavidin were conjugated using mAbcocktails that contained varying amounts of biotinylated isotype control(calculated as percentage CD3/CD28 mAb mass with CD3/CD28 ratio as 1:1).

FIG. 4. Comparison of T cell expansion and MACS bead expansion. Primaryhuman T cells were expanded over 2 weeks in well plates in variousculture conditions. (FIG. 4, Top) Day 14 brightfield images of T cellsexpanded at the indicated cell:carrier ratio with the absolute number ofcarriers held constant. (FIG. 4, Bottom) Day 14 fold change ofmicrocarrier cultures at indicated ratios compared with cells grown inMACSibeads culture (conventional magnetic beads).

FIG. 5 depicts undisturbed microcarrier cultures forming 3D cell:carrierclusters.

FIGS. 6A-6B. Assessment of memory subpopulations inmicrocarrier-expanded T cell cultures. T cells were expanded onmicrocarriers or MACSibeads cultures for 14 days and assessed via flowand transmigration assay. FIG. 6A) Expanded T cells assessed for CCR7and CD62L populations using flow cytometry. FIG. 6B) The same cellsassessed in (FIG. 6A) were characterized for functional migratorypotential.

FIGS. 7A-7B. Lentiviral transduction of microcarrier T cell cultures. Tcells were activated with either plate-bound antibodies or microcarriersand transduced with lentivirus expressing an anti-CD19 chimeric antigenreceptor. FIG. 7A) Flow cytometry plots of CAR-expressing T cellsassessed on day 9 of culture. FIG. 7B) Transduced T cells were assessedfor functionality by measuring degranulation in response to tumor cells.

FIGS. 8A-8B. Microcarrier T cell cultures across 3 donors. FIG. 8A) Foldchange and FIG. 8B) viability assessed at day 14 of culture at varyingcell:carrier ratios.

DETAILED DESCRIPTION OF THE DISCLOSURE

As specified in the Background Section, there is a great need in the artto identify technologies for improved methods and systems forlarge-scale production of suspension cells, such as for example and notlimitation, T cells, and use this understanding to develop novelbioprocessing systems and methods. The present disclosure satisfies thisand other needs. Embodiments of the present disclosure relate generallyto synthetic microenvironment capable of mimicking T cell niches withinsecondary lymphoid organs (such as for example and not limitation, lymphnodes) and more specifically to macroporous 3D microcarriers that can befunctionalized with antibodies to promote suspension cell (e.g., T cell)activation and expansion to mimic the environment found in lymph nodes.The systems should provide suspension cells with improved potency andefficacy and allow for specific sub populations to expand selectively.It is to such systems and methods of producing suspension cells, such asfor example and not limitation, T cells, that embodiments of the presentdisclosure are directed. T cells produced by the systems and methodsdescribed herein include, but are not limited to, recombinant T cells,gene modified T cells, chimeric antigen receptor (CAR) T cells,unmodified T cells, and CCR7⁺CD62⁺ central memory T cells.

Current approaches for CAR-based ACT are severely constrained by (a) thelimited availability of autologous T cells from cancer patients (b)difficulty in robustly and reproducibly expanding these cells to enoughnumbers for multiple administrations and (c) lack of quantifiablebiomarkers that are predictive for functional anti-cancer potency acrossvarious tumors. Current state-of-the-art methods involve culturingpatient-isolated T cells with α-CD3 antibodies (Ab) or α-CD3/α-CD28Ab-functionalized beads with high amounts of interleukins (ILs; such asfor example and not limitation, IL2, IL7 and/or IL15) in single cellsuspension. Even industry-based efforts have adopted this process withonly improvements being the use of (a) closed systems, e.g., bag-basedcultures and (b) rocking platforms (e.g., the Wave bioreactor). Thefield will benefit greatly from improved manufacturing processes forreproducible, rapid, more-efficient expansion of highly-potent T cells,with reduced cost.

Thus, new cell-manufacturing and biomarker characterization concepts,that would allow large scale production of therapeutic CAR-T cellswithout losing their potency, are critically needed. The inventorshypothesized that mimicking the cell-cell and autocrine/paracrinecommunication as well as the hypoxic microenvironment of the lymph node(LN) (where T cell expansion takes place in the body) along with modernbioreactor technologies would significantly enhance expansion of CAR-Tcells without loss in potency.

Herein is demonstrated the use of α-CD3/α-CD28-functionalizedmicrocarriers and cultured human T cells in LN-mimicking 3D niches whereT cells remain at high density with close cell-cell contact, and allowefficient paracrine/autocrine signaling. These parameters, absent fromcurrent T-cell manufacturing concepts, are likely critical since T cellssecrete large amounts of ILs locally to promote rapid, large scaleexpansion. Thus, the systems and methods described herein could alsoreduce culture media and IL requirements, thereby significantly reducingcost. Although microcarriers are primarily used for adherent cells,α-CD3/α-CD28 functionalization of microcarriers could allow T cells tobe anchored to the microcarrier 3D structure for improved activationand/or expansion. Effects of low oxygen tension, various cell-seedingdensities and α-CD3/α-CD28 ligand densities on expansion efficacy and Tcell quality were also studied. Methods according to the disclosure cancombine the LN-like niche with stirred tank or perfusion bioreactors, toaffect dynamic culture and flow perfusion and thus improve expansionefficacy (time and cell numbers), product quality, scalability and costeffectiveness. Further, porous microcarriers can mimic 3D LN-likeniches. Microcarriers are widely used for bioreactor cultures ofadherent cells (8-10), but not for non-adherent cells like T cells. Theuse of porous gelatin (denatured collagen) carriers with functionalizedanti-CD3/anti-CD28 would allow us to anchor T cells to the scaffolds,mimic the extracellular matrix (ECM) microenvironment of LN, and mimicthe APC/T-cell signaling events in a controlled manner. Porousmicrocarriers also provide a high surface area for culture and can beused with stirred tank and perfusion bioreactors to ensure large-scaleculture. Stirred tank bioreactors allow for easy scale-up of culturesand are widely investigated in cell bioprocessing (11-13). A singlebioreactor can replace a large number of static petri-dishes, canprovide a closed-culture system to reduce handling and contaminationduring manufacturing, eventually provide automated monitoring of cultureparameters (oxygen, pH, etc.) and can have better nutrient mixing.Porous microcarriers can be suspended in stirred tank bioreactors forrapid scale up.

Microcarrier cultures were originally developed for large-scale cultureof anchorage-dependent cells. Microcarriers allow for high density cellculture; typically, about two orders of magnitude higher cell densities(up to 2×10⁸ cells/mL, compared to 2-3 ×10⁶/mL cells withoutmicrocarrier) (18). This enables scaling up of cell manufacturingprocesses with smaller footprints with reduced overall consumption ofexpensive media, serum and growth factors. Porous microcarriers areparticularly suited for high density culture and significantly increasethe available surface area for cells. In addition, porous microcarriersare better protected against unwanted mechanical stress generated inbioreactors (11) However, microcarriers are typically always used foranchorage dependent cells (8-10). Herein it is shown that T cells arealso ideal candidates for porous microcarrier-based expansion because:(a) it allows high density culture similar to that inside LNs, thusproviding autocrine/paracrine IL signaling; (b) allows functionalizationof the carrier surface with anti-CD3/CD28, thus avoiding bead-basedsignaling. Specific, non-limiting embodiments described herein use theCultispher G microcarriers (Hyclone) due to several reasons: i) thismicrocarrier is made of gelatin, which is derived from collagen, one ofthe most abundant structural components of the LN ECM; ii) chemicalsurface modification is possible by use of unreacted amine or carboxylicacid groups in gelatin (as shown in U.S. Pat. No. 8,318,492); iii) uponcompletion of culture, close to 100% cell harvesting is possible bycomplete dissolution of gelatin matrix with enzymatic (e.g. trypsin)digestion; iv) highly crosslinked cavernous structure with high interiorsurface area allows greater number of cells (>2,000) per microcarrierthat are well protected from shear stress; and v) great mechanicalstability that allows for a long term culture.

To provide activation signals to CAR-T cells in 3-D, the surface of theporous microcarriers was modified with anti-CD3/anti-CD28 Abs in varyingdensities. In some non-limiting embodiments, sulfo-NHS-biotin (LifeTechnologies) was conjugated to the amine groups of gelatin followed byincubation with streptavidin to generate a fully streptavidin modifiedmicrocarrier. Streptavidin was thus a biolinker for further modificationwith biotinylated anti-CD3 and anti-CD28 Abs. By controlling therelative concentrations of biotinylated Abs, it was possible tosystematically vary the surface density of Abs. In other embodiments,amine- or carboxylate-reactive reagents (e.g., carbodiimide (EDC) orN-hydroxysulfosuccinimide (sulfo-NHS), Life Technologies) were used tofunctionalize the gelatin-based microcarriers with, for example and notlimitation, Protein A or G, which bind in high affinities to Fc regionof Abs with different number of binding sites per molecule (5 and 2,respectively). It was possible to vary the overall density ofanti-CD3/anti-CD28 Abs on the microcarrier surface by changingincubation concentrations, and also systemically vary the local valencyby changing using streptavidin, Protein A or Protein G.

Various bioreactors have been employed for scale-up cell manufacturingto enhance homogeneity of the system (including elements such asnutrients, cytokine/growth-factors, oxygen, cell density, etc.), improvesterility (closed system design), and augment biological functions(shear, flow) (19). Spinner flasks or stirred tank bioreactors (11-13)are the most-explored platform that supports expansion of a variety ofcell types including 3D cellular organizations (20). These bioreactorscreate a homogeneous physicochemical environment (19). However, thestirrer (or spinner) can apply irregular amount of mechanical shearforces on suspended cells and cell aggregates. Perfusion bioreactors, onthe other hand, minimizes such high shear, and can deliver freshnutrients and cytokines continuously while imposing fluid mechanicalforces on cells in a more controlled manner (21-24). In cultures withmicrocarriers, the high cell density requires frequent media exchanges,in which case the application of perfusion has significant benefit.Perfusion rates can be controlled to mimic the interstitial fluid flowregime (15), such as for example and not limitation, flow in the LNniches. In some embodiments of the present disclosure, stirred-tank typeambr™ micro-bioreactor systems (TAP Biosystems) that allows bioprocessoptimization at microscale (10-15 ml) are used, mimicking the corecharacteristics of classical bioreactors, but with reduced use of mediaand growth factors. Other embodiments can use the CartiGen perfusionbioreactor system (model C9-x, Instron), optionally without employingthe compression feature due to the unknown effects of the associatedmechanical stimuli. In either embodiment, fresh media can be perfusedusing a common flow loop in a varying flow rate, closely mimicking thephysiological interstitial flow rate, ranging from 0.1-2.0 μm/s 25.

Another major challenge for CAR-T cell manufacturing is that extensiveexpansion results in decreased potency, which is related to thedifferentiation status of T cells before and after the expansion. Thesystems and methods of the present disclosure result in improved potencyrelative to current methods of expansion.

Definitions

To facilitate an understanding of the principles and features of thevarious embodiments of the disclosure, various illustrative embodimentsare explained below. Although exemplary embodiments of the disclosureare explained in detail, it is to be understood that other embodimentsare contemplated. Accordingly, it is not intended that the disclosure islimited in its scope to the details of construction and arrangement ofcomponents set forth in the following description or examples. Thedisclosure is capable of other embodiments and of being practiced orcarried out in various ways. Also, in describing the exemplaryembodiments, specific terminology will be resorted to for the sake ofclarity.

It must also be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,reference to a component is intended also to include composition of aplurality of components. References to a composition containing “a”constituent is intended to include other constituents in addition to theone named. In other words, the terms “a,” “an,” and “the” do not denotea limitation of quantity, but rather denote the presence of “at leastone” of the referenced item.

As used herein, the term “and/or” may mean “and,” it may mean “or,” itmay mean “exclusive-or,” it may mean “one,” it may mean “some, but notall,” it may mean “neither,” and/or it may mean “both.” The term “or” isintended to mean an inclusive “or.”

Also, in describing the exemplary embodiments, terminology will beresorted to for the sake of clarity. It is intended that each termcontemplates its broadest meaning as understood by those skilled in theart and includes all technical equivalents which operate in a similarmanner to accomplish a similar purpose. It is to be understood thatembodiments of the disclosed technology may be practiced without thesespecific details. In other instances, well-known methods, structures,and techniques have not been shown in detail in order not to obscure anunderstanding of this description. References to “one embodiment,” “anembodiment,” “example embodiment,” “some embodiments,” “certainembodiments,” “various embodiments,” etc., indicate that theembodiment(s) of the disclosed technology so described may include aparticular feature, structure, or characteristic, but not everyembodiment necessarily includes the particular feature, structure, orcharacteristic. Further, repeated use of the phrase “in one embodiment”does not necessarily refer to the same embodiment, although it may.

Ranges may be expressed herein as from “about” or “approximately” or“substantially” one particular value and/or to “about” or“approximately” or “substantially” another particular value. When such arange is expressed, other exemplary embodiments include from the oneparticular value and/or to the other particular value. Further, the term“about” means within an acceptable error range for the particular valueas determined by one of ordinary skill in the art, which will depend inpart on how the value is measured or determined, i.e., the limitationsof the measurement system. For example, “about” can mean within anacceptable standard deviation, per the practice in the art.Alternatively, “about” can mean a range of up to ±20%, preferably up to±10%, more preferably up to ±5%, and more preferably still up to ±1% ofa given value. Alternatively, particularly with respect to biologicalsystems or processes, the term can mean within an order of magnitude,preferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated, theterm “about” is implicit and in this context means within an acceptableerror range for the particular value.

By “comprising” or “containing” or “including” is meant that at leastthe named compound, element, particle, or method step is present in thecomposition or article or method, but does not exclude the presence ofother compounds, materials, particles, method steps, even if the othersuch compounds, material, particles, method steps have the same functionas what is named.

Throughout this description, various components may be identified havingspecific values or parameters, however, these items are provided asexemplary embodiments. Indeed, the exemplary embodiments do not limitthe various aspects and concepts of the present disclosure as manycomparable parameters, sizes, ranges, and/or values may be implemented.The terms “first,” “second,” and the like, “primary,” “secondary,” andthe like, do not denote any order, quantity, or importance, but ratherare used to distinguish one element from another.

It is noted that terms like “specifically,” “preferably,” “typically,”“generally,” and “often” are not utilized herein to limit the scope ofthe claimed disclosure or to imply that certain features are critical,essential, or even important to the structure or function of the claimeddisclosure. Rather, these terms are merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the present disclosure. It is also noted thatterms like “substantially” and “about” are utilized herein to representthe inherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “50 mm” is intended to mean“about 50 mm.”

It is also to be understood that the mention of one or more method stepsdoes not preclude the presence of additional method steps or interveningmethod steps between those steps expressly identified. Similarly, it isalso to be understood that the mention of one or more components in acomposition does not preclude the presence of additional components thanthose expressly identified.

The materials described hereinafter as making up the various elements ofthe present disclosure are intended to be illustrative and notrestrictive. Many suitable materials that would perform the same or asimilar function as the materials described herein are intended to beembraced within the scope of the disclosure. Such other materials notdescribed herein can include, but are not limited to, materials that aredeveloped after the time of the development of the disclosure, forexample. Any dimensions listed in the various drawings are forillustrative purposes only and are not intended to be limiting. Otherdimensions and proportions are contemplated and intended to be includedwithin the scope of the disclosure.

As used herein, the term “subject” or “patient” refers to mammals andincludes, without limitation, human and veterinary animals. In apreferred embodiment, the subject is human.

In accordance with the present disclosure there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higginseds.(1985); Transcription and Translation (B. D. Hames & S. J. Higgins,eds. (1984); Animal Cell Culture (R. I. Freshney, ed. (1986);Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994); amongothers.

SYNTHETIC MICROENVIRONMENTS OF THE DISCLOSURE

The present disclosure provides a system for mimicking the secondarylymphoid organs where suspension cells (e.g., T cells) are expanded;methods of expanding, activating, and transfecting the suspension cellsin the synthetic microenvironment, and suspension cells produced by suchsystems and methods.

In one aspect, the disclosure provides a system for expanding,activating, and/or transfecting suspension cells comprising: a porousmicrocarrier; and the suspension cells.

In some embodiments, the suspension cells are isolated from a patient'sblood or organs, including both normal and/or diseased tissues.

In some embodiments, the suspension cells are T cells.

In other embodiments, the porous microcarrier is three dimensional. Insome embodiments, the porous microcarrier comprises proteins,carbohydrates, lipids or nucleic acids. In some embodiments, the porousmicrocarrier comprises gelatin or other extracellular matrix components.

In yet other embodiments, the porous microcarrier is functionalized. Insome embodiments, the functionalized porous microcarrier comprises atleast one of antibodies, aptamers, and phage-display identified peptideligands.

In some embodiments, the antibodies comprise antibodies that arespecific for the suspension cells. In other embodiments, the antibodiescomprise antibodies that are specific for T cells. In some embodiments,the antibodies comprise anti-CD2, anti-CD3 and/or anti-CD28 antibodies.

In an embodiment, the system in any of the preceding embodiments furthercomprises a bioreactor. In some embodiments, the bioreactor comprises aclosed bioreactor and an open bioreactor. In some embodiments, the openbioreactor comprises a static culture vessel with a gas-permeablebottom. In other embodiments, the closed bioreactor comprises astirred-type bioreactor, a bag bioreactor, and a perfusion bioreactor.

In another embodiment, the system in any of the preceding embodimentsfurther comprises at least one of culture medium, at least one cytokine,and/or at least one viral vector, and optionally at least one growthfactor.

In some embodiments, the at least one cytokine comprises IL2 andoptionally at least one of IL7 or IL15. In other embodiments, the atleast one viral vector is configured for use in gene therapy (such asfor example and not limitation, a lentiviral vector, a retroviralvector, an adenoviral vector, and an adeno-associated viral vector).

In any of the embodiments described herein, the suspension cells cancomprise recombinant T cells, gene modified T cells, chimeric antigenreceptor (CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ centralmemory T cells.

In some embodiments, the at least one viral vector comprises a CARtransgene and optionally at least one additional gene, wherein the atleast one additional gene comprises therapeutic genes, surface markergenes, reporter genes, suicide genes, chemokine receptor genes,cytokine-expressing genes, and/or immune-checkpoint receptor genes.

In any of the preceding embodiments, the porous microcarrier ismacroporous. In some embodiments, the porous microcarrier is degradable,such as for example and not limitation, biodegradable.

In a related aspect, the disclosure provides a method of expanding,activating, and/or transfecting suspension cells, the method comprising:obtaining a blood sample from a patient; isolating suspension cells fromthe blood sample; introducing the suspension cells to a bioreactorcomprising a porous microcarrier; activating the suspension cells;expanding the suspension cells; optionally transfecting the suspensioncells; preparing the suspension cells for transfusion into the patient;and transfusing the suspension cells into the patient.

In some embodiments, the blood or tissue sample is obtained byleukapharesis.

In other embodiments, the suspension cells are T cells.

In some embodiments, the step of isolating suspension cells from theblood sample further comprises bead separation or magnetic beadseparation.

In other embodiments, the bioreactor comprises a closed bioreactor andan open bioreactor. In some embodiments, the closed bioreactor comprisesa stirred-type bioreactor, a bag bioreactor, and a perfusion bioreactor.In other embodiments, the open bioreactor comprises a static culturevessel with a gas-permeable bottom.

In some embodiments, the porous microcarrier is three dimensional. Inother embodiments, the porous microcarrier comprises proteins,carbohydrates, lipids or nucleic acids. In still other embodiments, theporous microcarrier comprises gelatin or other extracellular matrixcomponents.

In some embodiments, the porous microcarrier is functionalized. In otherembodiments, the functionalized porous microcarrier comprises at leastone of antibodies, aptamers, and phage-display identified peptideligands. In still other embodiments, the antibodies comprise antibodiesthat are specific for the suspension cells. In some embodiments, theantibodies comprise antibodies that are specific for T cells. In someembodiments, the antibodies comprise anti-CD2, anti-CD3 and/or anti-CD28antibodies.

In an embodiment of any of the foregoing methods, the method furthercomprises at least one of culture medium, at least one cytokine, and/orat least one viral vector, and optionally at least one growth factor. Insome embodiments, the at least one cytokine comprises IL2, andoptionally at least one of IL7 or IL15.

In an embodiment of any of the foregoing methods, the activation stepfurther comprises agitation, optionally under hypoxic conditions. Insome embodiments, the agitation can be periodic or continuous. In otherembodiments, the activation step can last for at least one day, at leasttwo days, at least three days, and at least four days, and all ranges oftime in between.

In an embodiment of any of the foregoing methods, the expansion stepfurther comprises seed trains, optionally under hypoxic conditions. Forexample and not limitation, the expansion step can include increasingthe size of the flask and/or bioreactor as the culture grows, which canalso involve moving the expanding cells to a new vessel, and culturemedia exchanges every 2-3 days (particularly if a static culture). Theexpansion step can last for at least one day, at least two days, atleast three days, at least four days, at least five days, at least sixdays, at least seven days, at least eight days, at least nine days, atleast ten days, and at least eleven days, and all ranges of time inbetween.

In an embodiment of any of the foregoing methods, the optionaltransfection step further comprises adding at least one viral vector tothe bioreactor comprising the suspension cells.

In an embodiment of any of the foregoing methods, the suspension cellscomprise recombinant T cells, gene modified T cells, chimeric antigenreceptor (CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ centralmemory T cells.

In some embodiments, the at least one viral vector is configured for usein gene therapy. In other embodiments, the at least one viral vectorcomprises a CAR transgene and optionally at least one additional gene,wherein the at least one additional gene comprises therapeutic genes,surface marker genes, reporter genes, suicide genes, chemokine receptorgenes, cytokine-expressing genes, and/or immune-checkpoint receptorgenes.

In an embodiment of any of the foregoing methods, the preparation stepfurther comprises cell expansion and downstream bioprocessing. In someembodiments, the cell expansion and downstream bioprocessing comprisescell separation, purification, packaging, preservation, storage,shipping and transport, thawing, formulation, resuspension, andtransfusion.

In an embodiment of any of the foregoing methods, the transfusion stepfurther comprises injection, intravenous administration, andimplantation (such as for example and not limitation, implantation of asustained delivery device).

In an embodiment of any of the foregoing methods, the porousmicrocarrier is macroporous. In any of the preceding embodiments, theporous microcarrier is macroporous. In some embodiments, the porousmicrocarrier is degradable, such as for example and not limitation,biodegradable.

In a related aspect, the disclosure provides a T cell obtained from anyof the systems disclosed herein.

In a related aspect, the disclosure provides a T cell obtained from anyof the methods disclosed herein.

In one embodiment, the T cell is a memory cell. In another embodiment,the T cell is CCR7⁺CD62L⁺. In another embodiment, the T cell is acentral memory T cell. In yet another embodiment, the T cell is a CD4 Tcell or a CD8 T cell. In some embodiments, the T cell comprisesrecombinant T cells, gene modified T cells, chimeric antigen receptor(CAR) T cells, unmodified T cells, and/or CCR7⁺CD62⁺ central memory Tcells.

In a related aspect, the disclosure provides a composition comprising atleast one T cell as described herein. In some embodiments, thecomposition further comprises at least one carrier. In otherembodiments, the composition is formulated for intravenousadministration.

In a related aspect, the disclosure provides a pharmaceuticalcomposition comprising at least one T cell as described herein. In someembodiments, the pharmaceutical composition further comprises at leastone carrier. In other embodiments, the composition is formulated forintravenous administration. In some embodiments, the pharmaceuticalcomposition further comprises at least one additional therapeutic agent.

In a related aspect, the disclosure provides the use of a composition(including a pharmaceutical composition) as described herein to treat adisease or condition in a patient in need thereof. In some embodiments,the disease or condition comprises genetic diseases, cancers,infections, autoimmune diseases, and/or transplant complications. Insome embodiments, the use further comprises a second therapeutic methodor agent, such as for example and not limitation, a cancer drug, animmunotherapeutic, an immunosuppressant, an autoimmune therapeuticagent, and/or a therapeutic for treating infections.

In a related aspect, the disclosure provides method of treating adisease or condition in a patient in need thereof, comprisingadministering a composition (including a pharmaceutical composition) asdescribed herein to said patient. In some embodiments, the disease orcondition comprises genetic diseases, cancers, infections, autoimmunediseases, and/or transplant complications. In some embodiments, thetreatment further comprises a second therapeutic method or agent, suchas for example and not limitation, a cancer drug, an immunotherapeutic,an immunosuppressant, an autoimmune therapeutic agent, and/or atherapeutic for treating infections.

In an embodiment of any of the foregoing systems, the porousmicrocarrier is configured for activation, expansion, and/ortransfection of the suspension cells.

In an embodiment of any of the foregoing methods, the porousmicrocarrier is configured for activation, expansion, and/ortransfection of the suspension cells.

EXAMPLES

The present disclosure is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the disclosure or of any exemplified term.Likewise, the disclosure is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe disclosure may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the disclosure in spirit or in scope. The disclosure is thereforeto be limited only by the terms of the appended claims along with thefull scope of equivalents to which those claims are entitled.

Example 1 Development of the Synthetic Microcarrier System

Current T cell expansion technologies do not fully recapitulate thesecondary lymphoid organs where T cell are expanded with close cell-cellcontact under hypoxic conditions. Herein, functionalized microcarrierswere used in combination with modern bioreactors to create 3D nicheswhere T cells are stimulated to expand with anti-CD3 and anti-CD28antibodies while remaining in close cell-cell contact. The high surfacedensity of these microcarriers encouraged high cell density andefficient signaling, while cytokine requirements, media usage, andbioreactor footprint were reduced.

Development and Optimization of Antibody-Coated Microcarriers

Anti-CD3 and anti-CD28-coated microcarriers were generated byfunctionalizing the surface of Cultispher gelatin microcarriers by (1)conjugating sulfo-NHS-biotin to amine groups of gelatin, (2) incubatingwith streptavidin to create a fully streptavidin-coated microcarrier,and (3) adding biotinylated anti-CD3 and anti-CD28 antibodies atcontrolled densities to regulate the antibody surface density.

Cultispher-S (CuS) and Cultispher-G (CuG) are two types of microcarrierscommonly used in cell manufacturing and bioprocessing. They aregelatin-based and thus have many lysine residues that can befunctionalized using N-hydroxysuccinamide (NHS) chemistry. Thesecarriers were functionalized using streptavidin-biotin chemistry due tothe availability of biotinylated antibodies, the ease of use for thismethod, and the relative stability of this system. Sulfo-NHS-biotin wasthe chosen crosslinker for this system due to its solubility in waterand its relatively short spacer arm, which likely maximized theavailability of open streptavidin binding sites that can bind toantibodies.

First, the inventors quantified the amount of streptavidin that could bebound to the surface as a function of biotinylation (FIG. 1A).Microcarriers were first biotionylated using varying amountssulfo-NHS-biotin, and streptavidin was then introduced in excess toevenly coat the surface. Unreacted streptavidin from the supernatant wasmeasured using the BCA assay, which was then used in combination withthe estimated surface area of CuS and CuG microcarriers to determine thestreptavidin surface density as a function of the sulfo-NHS-biotinconcentration. It was assumed that the difference between the amount ofstreptavidin added to the suspension and the amount of streptavidinremaining in the supernatant was equal to the amount of boundstreptavidin. To make each curve in FIG. 1A, all samples were repeatedin duplicate and error bars in the chart represented standard deviationabout the mean. The curve fit was found using a modified Scatchard plotanalysis, which followed standard receptor-ligand kinetics and thusshowed saturation of the binding sites in a hyperbolic fashion. Themolecules per area was determined using the amount of streptavidinremaining in the supernatant and dividing by the surface area of themicrocarriers (mass of microcarriers was known and the manufacturersupplied the surface area per carrier mass. The estimated surface areasfor CuS and CuG microcarriers were 19,000 cm² and 24,000 cm²,respectively). Cultisper S has much higher binding efficiency, thereforethis microcarrier was used in all further experiments.

Excess biotin linked to the surface may negatively impact antibodyattachment efficiency, as it could theoretically block all availablebinding sites on the bound streptavidin molecules. As streptavidin hasfour binding sites, each molecule generally has a few sites occupied bybiotins attached to the surface of the microcarrier and the remainderare available to attach to biotinylated ligands such as antibodies; FIG.1B quantified the remaining open binding sites as a function ofbiotinylation. Microcarriers were coated as described herein and thenumber of available binding sites was quantified using a fluorimeterwith FITC-biotin as a surrogate ligand. FITC-biotin was added to thesuspension of streptavidin coated microcarriers and the supernatant wasassessed for fluorescence to quantify the amount of unbound FITC-biotinwhich was then used to find the amount of bound FITC-biotin (analogousto (FIG. 1A) above). One mole of FITC-biotin was assumed to be one moleof available binding sites. The area used for the calculation of bindingsites/um² was 19000 cm². Together, these show that approximately 5000nmol/g of sulfo-NHS-biotin is optimal for maximizing ligand surfacedensity, as this keeps the streptavidin surface density near saturation(and thus ensured an even coating throughout the carrier), while alsomaintaining a significant number of open binding sites.

FIG. 2 demonstrated a verification of open biotin binding sites viaZeiss Lightsheet microscope imaging of (A) uncoated CuS microcarriersand (B) CuS microcarriers optimally coated with streptavidin andsaturated with FITC-biotin. Laser power was fixed to 10% in both cases.Since the microcarriers were macroporous and thus could support cellgrowth on the interior, it was important to show that the conjugationstrategy as described could evenly and comprehensively coat the entiresurface of the microcarriers, including within the macropores on theinterior surface. FIG. 2 demonstrated this even coating because theinterior of the microcarriers appears comparably bright to the outersurface at a middle cross section, showing that we have coated the core.Furthermore, the uncoated sample rules out autofluorescence (backgroundsignal) which one might expect due to the fact that the carriers areprotein-based.

TABLE 1 Scale up of CuS microcarrier coating procedure. Anti-CD3 andanti-CD28 antibodies were added in a 1:1 ratio, and the Ab coatingprocedure was repeated for small, medium, and large batch sizes toverify process scalability. Parameters in medium and large batches werescaled proportionally to the small batch size. Microcarrier dry weightMedium Small (15 mg) (150 mg) Large (300 mg) Streptavidin/μm² 7500 85008200 Biotin-binding 1200 2700 1400 sites/μm² Antibodies/μm² 2600 30002600 Antibody:Streptavidin 0.35 0.35 0.31 Ratio

As shown in FIG. 3, microcarriers coated with streptavidin wereconjugated using monoclonal (mAb) cocktails that contained varyingamounts of biotinylated isotype control (calculated as percentageCD3/CD28 mAb mass with CD3/CD28 ratio as 1:1). This demonstrated thatthe isotype control mAbs and CD3/CD28 antibodies likely bind verysimilarly, thus showing that it is possible to control the amount ofactivating antibodies (CD3 and CD28) by spiking the cocktail used tocoat the microcarriers with a non-binding antibody (isotype control). Insummary, it is possible to fine-tune the activation signals delivered onthe microcarriers to expand T cells, which can make the microcarrierplatform more adaptable.

Variable Signaling Density on Microcarriers

An interesting parameter to vary is the signal strength delivered on thesurface of each microcarrier; this will require varying the number ofCD3 and CD28 antibodies conjugated to the carriers. To accomplish this,the inventors spiked the CD3/CD28 biotinylated mAb cocktail with abiotinylated isotype control (by definition, an antibody with unknowntarget) prior to adding the cocktail to the carriers for conjugation. Intheory, the isotype control should displace the available binding sitesfor the CD3/CD28 mAbs, effectively reducing the signal strength that canbe delivered per unit surface area. Signal strength was expressed as apercentage of full saturation. Microcarriers were coated at differentsignal strengths according to this method, and the total mAb density wasdetermined indirectly by measuring the supernatant with the BCA proteinassay. The slight downward slope of these data indicate that the bindingefficiency appears to be consistent, but the isotype control may have aslightly less binding capacity than the CD3/CD28 mAbs (possibly due todifferences in biotinylation capacity).

To test the effectiveness of the varied signal strength, the inventorsseeded primary human T cells on microcarriers with signal densitiesranging from 100% to 0% with 20% intervals. Cells were assessed on day 3for qualitative differences in activation potential (cell clusters, cellenlargement, media color changes). The media color ranged from yellow(100%) to pinkish red (0%) indicating a differential metabolic activitybetween the groups, which in turn was indicative of differentialactivation. This demonstrated that changing the signal density on themicrocarrier surface can have significant influence on activation state,and therefore likely can contribute to T cell expansion and phenotype. Tcell activation was further confirmed with the appearance of T cellclusters in the microcarriers.

Cell Seeding onto Antibody-Coated Microcarriers

The loading procedure for Ab-coated CuS microcarriers was optimizedusing Jurkat cells, an immortalized line of human T lymphocytes. Twoseeding methods were investigated, including a column seeding method andan extended orbital shaking method. The first procedure was designed topromote cell contact between cells and microcarriers in a syringe-basedcolumn. Briefly, the needle of a 0.5 mL syringe was removed using adremel, and the mesh from a 40 um cell strainer was wrapped around thebottom. The syringe/filter was placed in a FACS tube and loaded withAb-coated microcarriers, which were held in place by the cell strainermesh. Media was then added such that the liquid level inside and outsidethe syringe was the same. Finally, 1 million Jurkat cells werepre-labeled with CFSE fluorescent dye and added to the column, and theassembly was centrifuged for 3 cycles of 5 minutes at 300 g followed by10 minutes rest to promote attachment. Visualization of the cells withinthe microcarriers demonstrated that cell loading using this method washeterogeneous, with many microcarriers lacking cells. It washypothesized that the short length of time (˜30 min) may not besufficient to allow for cell attachment to microcarriers.

An alternative cell seeding method using overnight orbital shaking wasnext investigated. Anti-CD3-coated carriers were loaded into a 12-wellplate at a density of 12,000 microcarriers per well, to which 1 millionJurkat cells were added (83:1 cell to microcarrier ratio). The plateswere placed in an incubator and continuously agitated on an orbitalshaker at 60 rpm to promote cell attachment to the microcarriers. After14 hours of attachment, cells appeared to preferentially surround themicrocarriers. To directly quantify the degree of attachment, thecontents of each well were filtered through a cell strainer to separateunattached cells from microcarriers plus attached cells. Recoveredmicrocarriers were digested with dispase, which dissolved the carriersand allowed for cell recovery and direct quantification, whileunattached cells were quantified in the flow-through fraction.CD3-coated microcarriers achieved a greater percent cell attachmentcompared to non-coated microcarriers (11.2±2.1% vs. 3.9±0.3%), as wellas a concomitant decrease in the percent of unattached cells in theflow-through (73.2±8.4% vs. 92.5±1.6%), suggesting productive binding ofT cells to anti-CD3-coated microcarriers.

Expansion of Primary Human T Cells Using Ab-Coated Microcarriers

Having established preferential binding of Jurkat T cells toanti-CD3/CD28-coated microcarriers (albeit at low efficiency), it wasnext determined whether these functionalized microcarriers promoted theexpansion of primary human T cells. As a first step, T cell expansionwas verified using CD3/CD28-loaded magnetic beads, which is the currentstate-of-the-art method for expansion of patient-isolated T cells.First, T cells were isolated from cryopreserved human PBMCs using a MACSpan-T cell isolation kit and seeded with anti-CD3/CD28-loaded MACSiBeads(cell:bead ratio=1:2). Cultures were expanded over a period of 28 daysper manufacturer's instructions, which ultimately achieved a 50-foldexpansion with a high exogenous IL-2 concentration of 400 U/mL. Whenexogenous IL-2 was omitted, only a 7-fold expansion was achieved, whichis consistent with previous findings that IL-2 enhances T cellexpansion7. Further, T cells did not expand in the no-bead negativecontrol, confirming that CD3/CD28 stimulation is required for T cellexpansion.

Primary human T cells were expanded using either anti-CD3/CD28-coatedmicrocarriers or IgG negative control-coated microcarriers. As apositive control, T cells were also expanded using anti-CD3/CD28-loadedMACSiBead to compare degree of expansion. The process of seeding T cellsonto microcarriers was carried out as described (83:1 cell:microcarrierratio using overnight attachment on an orbital shaker), after whichcultures were removed from the orbital shaker and allowed to expand foran additional 6 days with periodic media exchanges. At this 7 daytimepoint, T cells cultured with anti-CD3/CD28-coated microcarriersexpanded and formed aggregates of cells adjacent to microcarriers, whileno such expansion occurred in cultures with IgG control-coatedmicrocarriers. Anti-CD3/CD28-coated microcarriers achieved a 2.5-foldexpansion over 7 days, which compared favorably with the 1.4-foldexpansion observed with the MACSiBead culture. Therefore, anti-CD3/CD28functionalized microcarriers were capable of stimulating primary human Tcell expansion.

To determine whether T cells were expanding within the interiormacropores of the microcarriers, microcarriers plus attached cells wereseparated from any unattached cells using a 40 um strainer, and cellswere quantified in both the flow-through fraction (unattached) or themicrocarrier-associated fraction (attached) as described previously. Amajority of the cells were recovered in the flow-through fraction, withonly 200,000 cells (7% of the total cell number) recovered in themicrocarrier-attached fraction, suggesting low cell attachment tomicrocarriers. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) staining of attached cells within isolated microcarriersconfirmed the low level of cell loading and further demonstrated theheterogeneity of cell loading across microcarriers. Indeed, a fewmicrocarriers had a dozen or more attached cells, while many more had nocell attachment at all. Therefore, it is likely that activation andexpansion of T cells is mostly occurring adjacent to the microcarriersrather than within the macropores.

FIG. 4 shows a comparison of T cell expansion and MACS bead expansion.Primary human T cells were expanded over 2 weeks in well plates invarious culture conditions. FIG. 4, top shows Day 14 brightfield imagesof T cells expanded at the indicated cell:carrier ratio with theabsolute number of carriers held constant. FIG. 4, bottom shows Day 14fold change of microcarrier cultures at indicated ratios compared withcells grown in MACSibeads culture (conventional magnetic beads).MACSibeads cells were expanded according to the manufacturer'sinstructions at 1:2 cells:beads with a cell density of 2.5e6 cells/ml.Fold change was assessed after day 14 by counting cells using anautomated cell counter. The data show that in the case of microcarrierculture there is an optimal cell:carrier ratio where the fold changeoutperforms conventional magnetic bead culture.

When left undisturbed over the course of 14 day culture, T cells andmicrocarriers will form clusters as shown in FIG. 5. As shown in FIG. 5,the microcarriers (approximately 10, seen as dark ovals) were pulledtogether by T cells (fuzzy grey clusters on the periphery of themicrocarriers). These findings demonstrated that the microcarriers canfacilitate cell:carrier adhesion and high cell density. High celldensity is a key characteristic found in human lymph nodes that theinvented system is configured to mimic.

Process Optimization of Microcarrier-Based Expansion

The inventors then sought to determine the effects of IL-2 and celldensity on the expansion of T cells on microcarriers. To this end, theinventors created a 2×2 design matrix with high and low IL-2 and celldensity levels. The levels for IL-2 were 50 and 400 U/ml, respectively,and the levels for cell density were 1e4/ml and 2.5e6/ml. Expansion andphenotype were evaluated after day 14. It was observed that within theseconditions, only the higher density cell cultures expanded (the lowerlevel did not expand at any measurable level). Maximal expansion canvary as a function of cell:carrier ratio depending on the donor. Betweenthe two groups that did expand, IL-2 did not appear to affect expansionbut may have influenced memory phenotype, as assessed using flowcytometry and markers CD62L, CCR7, and CD45RA, resulting in a higherpopulation of central memory T cells (Tcm), including CD4 and CD8central memory T cells.

The inventors then further characterized the memory T cellsubpopulations expanded using the invented microcarrier system. As shownin FIG. 6, T cells were expanded on microcarriers or MACSibeads culturesfor 14 days and assessed via flow and transmigration assay. FIG. 6A)Expanded T cells were assessed for CCR7 and CD62L populations using flowcytometry. T cells that are CCR7⁺CD62L⁺ are memory T cells that home tothe lymph nodes and represent a population of T cells that have enhancedproliferative and migratory potential compared to effector T cells(CCR7⁻CD82L⁻). The qualities are important for cell immunotherapiesbecause highly proliferative cells can divide more in response to tumorantigen encounter, thus increasing efficacy of the therapy. Migratorycapacity is also important because migratory cells can travel to morelocations throughout the body (especially the lymph nodes where tumorantigens may be encountered) and will last longer, providing long-livedprotection. The data show that compared to MACSibeads cultures, the useof microcarriers in the invented system led to a higher frequency ofCCR7⁺CD62L⁺ phenotypes. Thus, the invented system comprisingmicrocarriers can produce higher quality T cells for immunotherapy. FIG.6B) The same cells assessed in (FIG. 6A) were characterized forfunctional migratory potential. CCR7 is a receptor for the chemokineCCL21; binding of CCL21 to CCR7 will thus trigger a T cell to migration.When T cells were incubated in a transwell with CCL21 on the oppositeside of a membrane, it was observed that more T cells crossed themembrane in the case of microcarrier-expanded T cells than MACSibeadsexpanded T cells. This confirmed the observations from (FIG. 6A) bydemonstrating that the increased number of CCR7⁺CD62L⁺ T cellscorrelates with increased migration.

Lentiviral Transduction of Microcarrier T Cell Cultures

T cells were activated with either plate-bound antibodies ormicrocarriers and transduced with lentivirus expressing an anti-CD19chimeric antigen receptor. FIG. 7A shows flow cytometry plots ofCAR-expressing T cells assessed on day 9 of culture. CAR was labelledusing biotinylated Protein L with APC-conjugated streptavidin assecondary. Plate-bound antibody stimulation is a conventional, smallscale technique for T cell activation. T cells must be activated to betransduced by a lentivirus. These data demonstrated that microcarriers,while fundamentally different from widely used standards such asplate-bound activation, can still provide a strong enough activationsignal for T cells to be transduced at similar levels. FIG. 7B shows thefunctionality of transduced T cells by measuring degranulation inresponse to tumor cells. Degranulation was assessed in unstimulated(neg), K562 coculature (CD19⁻), K562⁻ CD19⁺ coculture (CD19⁺), andanti-CD3-stimulated (pos) groups and was measured using flow cytometryand quantifying surface expression of CD107a. Degranulation of T cellsis a hallmark of T cell cytotoxocity, which is important in assessingtheir ability to kill tumors. The model tumor cells in this case wereK562 cells, and the target the T cells were programmed to attack wasCD19. K562s are naturally do not express CD19 (the CD19⁻ group in thechart) so a genetically engineered K562 line with CD19 inserted (CD19⁺)was utilized as a target with the ligand that the CAR T cells shouldrecognize. This was also compared to a non-stimulated control, whichshould produce no degranulation, and a fully stimulated positive controlusing antibodies to trigger degranulation. Each group was compared usingan ANOVA and Control Dunnett's test with the control as the “neg” groupand alpha of 0.05. In FIG. 7B, the circles to the right representsignificance; non-overlapping circles indicate that groups are verysignificantly different. The data demonstrate that the in bothplate-bound and microcarrier-activated groups, T cells showed elevateddegranulation when presented with either CD19⁺ target cells or whenstimulated with antibodies. The fact that the T cells did not showelevated degranulation in the CD19⁻ group demonstrated that the CAR Tcells did not recognize any other proteins on the K562 cells, ruling outoff-target effects. In summary, these data show that T cells transducedusing microcarriers are functionally equivalent to a widely usedstandard.

Microcarrier T Cell Cultures Across Multiple Donors

As shown herein, microcarriers can induce expansion of T cells, and thisexpansion may be superior to conventional techniques (such as MACS) interms of expansion capacity. The inventors next asked if this techniquecould work across multiple donors. The invented microcarrier system wastested at varying cell:carrier ratios (80:1 to 10:1) with constantcarrier/well using T cells from 3 healthy donors (aged 25, 47, and 48)(FIG. 8). All donors showed similar trends, with lower cell:carrierratios showing higher expansion. Furthermore, all groups showedsimilarly high viability (with the exception of one). Cell expansionroughly correlated with age, with the youngest donor (number 3)expanding the greatest in the majority of groups and the oldest(donor 1) expanding the least. This could be due to metabolicdysfunction that accumulates with age. Fold change (FIG. 8A) andviability (FIG. 8B) were assessed at day 14 of culture at varyingcell:carrier ratios. These results demonstrated that the functionalizedmicrocarriers showed the same trend across different donors, thusshowing broad applicability. However, the exact behavior depends on eachspecific donor, with younger donors (donors 2 and 3) generally showingbetter expansion than the older donor (donor 1). Viability in most caseswas also very high across all donors, which further demonstrates thatthe invented systems and methods are robust.

Materials and Methods Microcarrier Functionalization

Cultispher S and Cultipher G microcarriers (HyClone) were suspended in1× PBS at 15 mg/ml and autoclaved at 121° C. for 15 minutes. Allsubsequent steps were performed in a sterile environment. Carriers werebiotinylated by adding 0.5 μl 10 mM sulfo-NHS-biotin (Thermo Fisher) permg microcarriers to the suspension and vortexing continuously for 60minutes at room temperature. Excess reagent was removed by washing thecarriers 3 times with 1× PBS by diluting 15-fold. Streptavidin (JacksonImmunoResearch) was added to the carrier suspension at 40 μg/ml andvortexed for 45 minutes. Supernatent samples for streptavidinquantification were taken after this step, and this was followed by 2wash steps in PBS by diluting 15-fold. For antibody coating,biotinylated low endotoxin, azide free (LEAF) anti-CD3, anti-CD28, ormouse IgG1 isotype controls (Biolegend) were added at predefinedmixtures to make a total antibody concentration of 30 μg/ml and vortexedfor 60 minutes. The anti-CD3 and anti-CD28 ratio was always kept 1:1.Mouse IgG1 isotype control was added to some microcarriers to displacethe anti-CD3/anti-CD28 antibodies for purposes of lowering the signalstrength presented on the surface. Antibody-coated microcarriers werewashed 2 times with 1× PBS by diluting 15-fold. Prior to cell culture, 1ml of microcarriers at 15-20 mg/ml was washed with 9 ml of appropriatemedia. Quantification of microcarriers was determined at the end of theprocess by sampling the microcarriers into a 96 well plate and countingmanually.

Streptavidin Binding Quantification

Streptavidin binding was quantified indirectly by measuring unboundstreptavidin in the supernatant immediately after the streptavidinconjugation and incubation steps. Streptavidin protein concentration wasmeasured using a BCA assay (Thermo Fisher) according to themanufacturer's instructions with several modifications. Briefly, a log 2standard curve was created starting at 40 μg/ml streptavidin (JacksonImmunoResearch). The assay was performed in a TC-treated 96 well plate.All sample or standard volumes were 150 ul, and the added BCA reagentwas 150 ul. The reaction was allowed to proceed in a dry incubator at37° C. for 45-90 minutes prior to reading at 562 nm on a Biotek PlateReader.

Microcarrier Binding Site Quantification

The number of open streptavidin binding sites was indirectly quantifiedby measuring the supernatant following FITC-biotin incubation.Immediately after washing excess streptavidin, microcarriers weresuspended at 15 mg/ml, and 80 μl of 5 μM FITC-biotin (Thermo Fisher) wasadded to the carrier suspension and vortexed for 20 minutes. The carriersuspension was centrifuged at 4500 g for 1 min to pull carriers to thebottom, and a 200 μl sample was assayed against a standard curve toquantify the unbound FITC-biotin.

Lightsheet Imaging of Functionalized Microcarriers

Binding site uniformity was confirmed qualitatively using a ZeissLightsheet microscope. Microcarriers were coated with streptavidin asdescribed earlier and all binding sites were saturated with FITC-biotin.Microcarriers were then resuspended in 0.1% agarose heated to 70° C. andcast into a thin capillary. Streptavidin coated microcarriers withoutFITC-biotin were used as negative control for autofluorescence.

Microcarrier and MACSiBeads Cell Culture

Primary human T cells were obtained from cryopreserved peripheral bloodmononuclear cells (PBMCs) (Zenbio) after separation with a negativeselection magnetic activated cell sorting kit (Miltenyi Biotech).MACSibeads cell culture was performed according to the manufacturer'sinstructions (Miltenyi Biotek). Briefly, magnetic MACSibeads wereconjugated with the provided anti-CD3 and anti-CD28 antibodies. Beadswere added to the T cell cultures at a 1:2 bead:cell ratio, and initialcell density was 2.5e6/ml in 96 well plates with total volume of 300 ul.Microcarrier cultures were performed by adding 45 mg (approximately36,000) microcarriers/well in 12 well plates and adding T cells incell:bead ratios of 83, 25, or 10 with final media volume of 2 ml.

Cultures were allowed to expand for 14 days, after which they wereassessed for fold change and phenotype via flow cytometry and chemotaxisassay. Fold change was quantified using a Countess automatic cellcounter (Thermo Fisher). Media was added after day 3 every 1-2 daysbased on media color. In all cases, recombinant human IL2 (Peprotech)was added to media at 400 U/ml. Media in all cases was either RPMI-1640(Thermo Fisher)+10% FBS (Hyclone) or OpTmizer with T cell expansionsupplement (Thermo Fisher).

Viral Transduction

T cells were transduced using RetroNectin (Thermo Fisher) following theplate-coating protocol provided by the manufacturer. Briefly, 50 μl of50 μg/ml working solution of Rectronectin was added to a non-TC-treated96 well plate. The plate was sealed and incubated overnight at 4° C. Theplate was then washed and blocked with 2% BSA solution. After BSA wasaspirated, plate was stored at 4° C. until future use.

For plate-bound antibody T cell cultures, the plate was prepared byadding a working solution of anti-CD3 and anti-CD28 low endotoxin, azidefree (LEAF) antibodies (Biolegend) at 1 ug/ml and 2 ug/ml respectivelyto a non-TC-treated 96 well plate. Plate was incubated overnight at 4°C. and washed twice with PBS prior to culture. T cells were added toeach well at 2.5e6 cell/ml with 300 μl total media per well (OpTmizer,Thermo Fisher). Microcarrier cultures were carried out as described.

Cells were transduced on day 1 of culture using a VSV-G pseudotypedlentivirus with the αCD19-41BB-CD3ζ (30) chimeric antigen receptor asthe genetic payload (Emory Viral Vector Core). In retronectin plates,each coated well was filled with 50 ul media and appropriate amount ofvirus to achieve an MOI of 5 or 15 based on starting cell number. Wellswere mixed well using a micropipette to ensure even coating andcentrifuged for 2 hours at 32° C. and 2000 g. Media was removed fromretronectin wells, and T cells were transferred to the retronectin plateto begin transfection. In the case of microcarrier samples, themicrocarriers were resuspended well and transferred to the retronectinplate along with the cells. Transfection was allowed to proceed for 24hours, after which cells were transferred again to a fresh plate whereculture continued until day 14 with normal media additions. Eachexperimental group was performed in triplicate.

Flow Cytometry for Memory Cell Populations

Live cells were stained in flow buffer (PBS containing 0.5% BSA and 2 mMEDTA) at a density of 100,000 cells per 100 μL. Antibodies were added ata 1:20 dilution and incubated for 2 hours at 4 oC protected from light(anti-CD3-APC-H7, anti-CD4-PerCP-Cy5.5, anti-CD45RA-FITC,anti-CCR7-Alexa 647, anti-CD62L-PE, BD Biosciences). Cells weresubsequently washed in flow buffer and analyzed using a BD LSRFortessa.The data were analyzed using FlowJo software.

Chemotaxis

T cells were resuspended in RPMI medium (Life Technologies) containing0.2% BSA (Sigma) at a density of 300,000 cells per 100 μL, and 100 μI ofthis cell suspension was added to the apical side of 24-well Transwellfilters (Costar, 5 μm pore size). The chemokine CCL21 (PeproTech) wasadded to the basolateral chamber of the Transwells at a concentration of0, 250, or 1000 ng/mL in RPMI+0.2% BSA. After loading, Transwells wereplaced at 37° C., and the cells were allowed to migrate for 4 hours.Cells were then collected from the basolateral chamber and quantifiedvia CountBright beads (Thermo Fisher) using an Accuri C6 flow cytometer.The data were analyzed using FlowJo software.

Quantification of CAR Expression Using Protein L

CAR expression was quantified as described previously (38). Briefly, atleast 1e5 cells were transferred to flow tubes. Cells were washed threetimes with FACS buffer (PBS with 2% bovine serum albumin and 5 mM EDTA).1 μl Protein L (Thermo Fisher) at 1 mg/ml was added to each sample.Following incubation for 45 minutes, Protein L was washed out 3 threetimes with FACS buffer. In all samples 500 ng/ml streptavidin-PEconjugate was added and allowed to bind for 45 minutes. Cells were thenwashed again three times with FACS buffer. Cells were analyzed on a BDAccuri and events were quantified using FlowJo software. The positivegate was set at the boundary of an untransduced population.

Degranulation Assay

CD8+ T cell degranulation was assayed as previously described (6).Briefly, after 14 days of expansion, T cells from each group werepooled, and 1e5 were added to a V-bottom 96 well plate in OpTmizer mediawith T cell expansion supplement. Each well received a stimulationcocktail of anti-CD49d (eBioscience), anti-CD28 (Biolegend) and monensin(Thermo Fisher) at concentrations of 1 μg/ml, 1 μg/ml, and 2 μMrespectively. Positive control wells received an additional stimulationof 5 μg/ml anti-CD3 (Biolegend). Tumor cell wells received eitherwild-type K562 cells or CD19-transduced K562 cells (each at 1e5/well).Negative control wells received only media. The plate was centrifuged at100 g for 1 minute and incubated for 4 hours at 37° C.

Data was analyzed via flow cytometry by staining for CD107a and CD4 andquantifying the CD107a+ fraction of the CD4− population. T cells weredistinguished from K562 cells via FSC/SSC. All data was quantified usingFlowJo Software.

While several possible embodiments are disclosed above, embodiments ofthe present disclosure are not so limited. These exemplary embodimentsare not intended to be exhaustive or to unnecessarily limit the scope ofthe disclosure, but instead were chosen and described in order toexplain the principles of the present disclosure so that others skilledin the art may practice the disclosure. Indeed, various modifications ofthe disclosure in addition to those described herein will becomeapparent to those skilled in the art from the foregoing description.Such modifications are intended to fall within the scope of the appendedclaims. The embodiments of the present invention are also not limited tothe particular formulations, process steps, and materials disclosedherein as such formulations, process steps, and materials may varysomewhat. Further, the terminology employed herein is used for thepurpose of describing exemplary embodiments only and the terminology isnot intended to be limiting since the scope of the various embodimentsof the present invention will be limited only by the appended claims andequivalents thereof.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

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1. A system for expanding, activating, and/or transfecting suspensioncells comprising: a three-dimensional functionalized porousmicrocarrier; and suspension cells.
 2. (canceled)
 3. The system of claim1, wherein the suspension cells are T cells. 4.-7. (canceled)
 8. Thesystem of claim 1, wherein the functionalized porous microcarriercomprises at least one of antibodies, aptamers, and phage-displayidentified peptide ligands.
 9. The system of claim 1, wherein thefunctionalized porous microcarrier comprises antibodies that arespecific for the suspension cells.
 10. The system of claim 3, whereinthe functionalized porous microcarrier comprises antibodies that arespecific for T cells.
 11. The system of claim 1, wherein thefunctionalized porous microcarrier comprises one or more of anti-CD2,anti-CD3 and anti-CD28 antibodies. 12.-13. (canceled)
 14. The system ofclaim 1 further comprising an open bioreactor comprising a staticculture vessel with a gas-permeable bottom.
 15. The system of claim 1further comprising a closed bioreactor selected from the groupconsisting of a stirred-type bioreactor, a bag bioreactor, a perfusionbioreactor, and combinations thereof.
 16. A system for expanding,activating, and/or transfecting T cells comprising: a three-dimensionalfunctionalized macroporous microcarrier; T cells; and at least one of: aculture medium; a cytokine; a viral vector; and a growth factor.
 17. Thesystem of claim 16 comprising at least the cytokine selected from thegroup consisting of IL2, IL7, IL15, and combinations thereof.
 18. Thesystem of claim 16, wherein the T cells are selected from the groupconsisting of recombinant T cells, gene modified T cells, chimericantigen receptor (CAR) T cells, unmodified T cells, CCR7+CD62+ centralmemory T cells, and combinations thereof.
 19. The system of claim 16comprising at least the viral vector that is configured for use in genetherapy.
 20. The system of claim 16 comprising at least the viral vectorcomprising a CAR transgene, the system further comprising an additionalgene selected from the group consisting of a therapeutic gene, surfacemarker gene, reporter gene, suicide genes, chemokine receptor gene,cytokine-expressing gene, immune-checkpoint receptor gene, andcombinations thereof.
 21. (canceled)
 22. A method comprising: obtaininga blood sample from a patient; isolating suspension cells from the bloodsample; introducing at least a portion of the suspension cells to abioreactor comprising a three-dimensional functionalized porousmicrocarrier; activating at least a portion of the suspension cellsintroduced into the bioreactor; and expanding at least a portion of theactivated suspension cells. 23.-28. (canceled)
 29. The method of claim22, wherein the porous microcarrier comprises one or more of proteins,carbohydrates, lipids and nucleic acids. 30.-31. (canceled)
 32. Themethod of claim 22, wherein the functionalized porous microcarriercomprises at least one of antibodies, aptamers, and phage-displayidentified peptide ligands. 33.-34. (canceled)
 35. The method of claim22, wherein the functionalized porous microcarrier comprises one or moreof anti-CD2, anti-CD3 and anti-CD28 antibodies. 36.-47. (canceled) 48.(canceled)
 49. A suspension cell obtained by the method of claim 22.50.-56. (canceled)
 57. A pharmaceutical composition comprising: at leastone suspension cell of claim 49; at least one carrier; and at least oneadditional therapeutic agent; wherein the composition is formulated forintravenous administration. 58.-63. (canceled)
 64. The method of ofclaim 22 further comprising: transfecting at least a portion of theexpanded suspension cells; preparing at least a portion of thetransfected suspension cells for transfusion into the patient; andtransfusing at least a portion of the prepared suspension cells into thepatient; wherein the porous microcarrier is configured for activation,expansion, and/or transfection of the suspension cells. 65.-68.(canceled)